Summary of Work: The embryonic and early postnatal mammalian central nervous system (CNS) dynamically changes in cell number and anatomical structure as cells proliferate, undergo apoptosis, migrate and differentiate. This project line complements research conducted in project Z01 NS 02019-26 (Physiological properties developing on CNS cells), most of which involves direct recording of cell physiology in real time (electrophysiology), or near real-time calcium imaging. The long-term goal involves explanation of specific steps in CNS morphogenesis. This includes: 1) immunocytochemical study of GABAergic components in vivo using thin tissue sections; 2) quantitative analysis of properties emerging in vivo using flow cytometry; and 3) migration/motility studies in vitro of neuroblast precursors forming the embryonic cortex. We have discovered that virtually all cells throughout the entire embryonic CNS transiently express surface epitopes, which can be detected using tetanus toxin and A2B5. We used flow cytometry to profile specific membrane and cytoplasmic properties in epitope-identified proliferating precursors and differentiating cells. At the peak of neurogenesis, specific GABA(A) receptor subunits were found in the ventricular/subventricular zone (VZ/SVZ) (alpha 4, beta 1, beta 2, and gamma 1), while others (alpha 3, beta 3, and gamma 2) were primarily detected in the cortical plate/subplate (CP/SP) region. Since many cortical cells are themselves GABA-immunopositive, GABA may be interacting with these receptors. Flow cytometry was used to profile emergent GABA-related components. Surface epitope expressions correlated closely with pre- and postmitotic stages of neuronal and glial lineage progressions in cortical dissociates. Seven major subpopulations were immunoidentified in the prenatal cortex. GABA-related components (GAD 65, GAD 67, GABA, GABA(A) receptor subunits) and GABAergic signals (cell depolarization, elevated cytosolic cytosolic calcium levels) were almost exclusively confined to pre- and postmitotic cells expressing tetanus toxin binding, thereby characterizing them as neuronal. Subunit expressions of immunoidentified neuronal precursors paralleled their localization in cells composing the VZ/SVZ, while subunits expressed by tetanus toxin-positive neuronal populations corresponded to subunits localized to cells in the CP/SP region of tissue sections. Anti-GABA immunostaining of live cortical cells and growth cones studied with flow cytometry revealed surface expression of GABA immunoreactivity whose intensity was directly related to tetanus toxin binding levels over ~100-fold ranges in both signals. To test the hypothesis that GABA interacted directly with a tetanus toxin target, liposomes were doped with a toxin target, the ganlgioside GT1b. The FACS results indicate that GT1b complexes GABA and strongly suggest that GT1b and GABA co- localize on neuronal surfaces. Confocal microscopy of GABA and GT1b expression of cultured neurons showed that the two signals were co-localized. GABAergic properties were critical to neurite outgrowth in cultured neurons since experimental manipulations of GABA synthesis or GABAergic signals all profoundly affected process formation, if not cell survival. Chemoattractant roles for GABA in rat cortical neuroblast migration were studied using chemotaxis assays of dissociated cells and slice cultures. Neuroblasts dissociated from the VZ/SVZ migrated in a gradient-dependent manner (chemotaxis) to femtomolar GABA (10-15M), while CP/SP neurons exhibited gradient- independent motility (chemokinesis) in response to micromolar GABA. These effects were mimicked by structural analogues of GABA and blocked by preventing fluctuations in cytosolic calcium. CP/SP neurons, most of which were GABAergic (GAD 65+, GAD67+, GABA+), became spontaneously motile in the presence of bicuculline, a competitive antagonist of GABA at GABA (A) receptor/chloride channels. All of the GABAergic migration and much of the motility was blocked by pertussis toxin, implicating GTP-binding proteins. Collectively, the results reveal complex and apparently opposing roles for GABA in the movement of GABAergic cortical neurons. Physiological elevations in extracellular potassium (7-20mM) also blocked GABA-induced migration and motility. The results have been reproduced using anatomically-intact slice cultures, indicating that endogenous GABA is a chemoattractant that functions during cortical morphogenesis. Parallel experiments on chemotropism were carried out in normal and trisomy 16 mice, a model of Down Syndrome. Surprisingly, glutamate rather than GABA was the primary chemoattractant. Glutamate?s motility signals occurred via N- methyl-D-aspartate (NMDA)-type receptors. Considerably fewer neurons migrated in response to NMDA in trisomy 16 mice, while comparable numbers migrated to GABA. In sum, these results indicate that different amino acids in rat and mouse play primary roles in migration and motility and cortical neuronal migration failure occurs during morphogenesis of the cortex in a model of Down Syndrome.